Introduction
Cell proliferation is the most fundamental phenotypic property of cancer. The stimulus for cellular proliferation is central not only at the late steps in carcinogenesis, the cancer, but also at the earliest known step, initiation (1,2) and FIG. 1. In fact, cell proliferation exerts an influence in the initiation of carcinogenesis in that cells in the S phase are more sensitive toward many initiators than at other times in the cell cycle (3). A myriad of short-term tests exist for the assessment of the carcinogenic potential of chemicals. These tests detect only carcinogens that interact with nucleic acids, or induce DNA repair synthesis or mutations in bacterial or mammalian cells (4-8).
As testing of the genotoxicity and carcinogenicity of chemicals has become routine, a growing number of compounds have been found to induce tumors in chronic bioassays while exhibiting negative results in genotoxicity tests (9). Significant examples of these classes of compounds include the dioxins, chlorinated biphenyls and peroxisome proliferators. These chemicals are often active as tumor promoters in two-stage experiments and exhibit biological activities as hormones (ethinylestradiol), peroxisome proliferators (pirnixic acid) or enzyme inducers (phenobarbital) (10).
At the present time only the initiation-promotion assay is employed routinely. In this assay the test compounds are examined for their ability to promote hepatic tumors or foci formation after initiation with a known genotoxic agent (11,12). As currently formatted, this assay utilizes animals, requires several months to perform, and produces histological endpoints that are difficult to quantify and do not lend to rigorous dose-response calculations for the purposes of risk assessment (13).
Stimulation of DNA synthesis has been proposed as an assay for short-term assessment of nongenotoxic carcinogens and tumor promoters in vivo (14,15). This methodology has potential for application to routine testing. So far, only one result has been detected that is inconsistent with carcinogenicity bioassay data. The different carcinogenicity of di(2-ethylhexyl)adipate (negative in rats) and di(2-ethylhexyl)phthalate (positive) was not detectable by DNA stimulation index using .sup.3 H-thymidine. Both plasticizers were positive in this short-term system with doubling doses of 0.7 mmol/kg for di(2-ethylhexyl)adipate and 0.5 mmol/kg for di(2-ethylhexyl)phthalate. Other disadvantages of this system include the use of radioactivity and the high coefficient of variation in the endpoint.
Several in vitro models have been utilized for the assessment of nongenotoxic carcinogens. Chida et al. (16) modeled the activation of protein kinase C and specific phosphorylation of a 90,000 kDa membrane protein of promotable BALB/3T3 and C3H/10T1/2 cells by tumor promoters. Smith and Colburn also utilized protein kinase C and its substrates in tumor promoter-sensitive and tumor-resistant cells as a biochemical marker for the response of cells to tumor promoters (17). However, these systems were flawed by both false positive and false negative values. The false positive values may be due to the fact that the activation of protein kinases C represents a biochemical signal far upstream from the final proliferative signal, while the false negatives may result from the fact that protein kinase C represents only a single receptor-mediated response. At least four other receptor responses, which are independent of protein kinase C, are known for tumor promotion and activity of nongenotoxic carcinogens (e.g. dioxin receptor, peroxisome proliferator receptor, phenobarbital receptor and estrogen receptor) (14,18).
Protein tyrosine phosphorylation
Protein-tyrosine kinases (PTK) constitute a class of enzymes that catalyze the transfer of the .mu.-phosphate of either ATP or GTP to specific tyrosine residues in certain protein substrates. Evidence suggests that these enzymes are important mediators of normal cellular signal transduction (19-21), with PTK being the intracellular effectors for many growth hormone receptors (22-24). PTK are also frequently the products of proto-oncogenes (25) and their aberrant expression has been associated with a variety of human cancers (26).
The cascade of protein tyrosine phosphorylation following the activation of protein tyrosine kinases appears to regulate the proliferative response (27,28). Specific, protein tyrosylphosphorylations are common to a wide variety of nongenotoxic carcinogens independent of associated receptors or known mechanism of action. The present invention demonstrates the xenobiotic alterations in protein tyrosine phosphorylation at a fundamental point in the control of cellular proliferation and on an assay protocol that characterizes the ability of a xenobiotic test chemical to initiate cellular proliferation.
Cyclin-dependent Kinases (CDK)
Recent experimental evidence suggests that the cell cycle of all eukaryotic cells is controlled at several checkpoints by different members of a novel class of protein kinase, the cyclin-dependent kinases (29, 31, 36, 46). The most well known of these kinases is the 34 kD product of the cdc2 gene in the fission yeast p34.sup.cdc2 ; however, several putative cyclin-dependent kinases (CDK) have now been cloned or identified. Some of these clones resemble p34.sup.cdc2.
At least nine CDKs have been described in the literature; these all have a common PSTAIR (SEQ. ID. NO:1) epitope. Therefore anti-PSTAIR would be expected to cross react with the entire complement of CDKs showing up in the 32 to 34 kD region. (Apparently some cyclins also cross react with the anti-PSTAIR antibody and this explains the banding at approximately 60 kD observed in some of the immunoblots with anti-PSTAIR.)
The antibody to the C-terminus region is more specific for p34.sup.cdc2 kinase, since the C-terminus region is more variable than the highly conserved PSTAIR (SEQ.ID.NO:1) region. However, it is obviously not species-specific since it was generated against human cdc2 and it cross reacts with mouse, rat and dog p34.sup.cdc 2 kinase.